The human DNA repair protein 0-6 methylguanine DNA methyltransferase (MGMT) is a major determinant in the sensitivity of tumors to chemotherapeutic chloroethylnitrosoureas (CENU). The long term objective of this proposal is to understand how MGMT interacts with lesioned substrates as a basis for the rational design of novel strategies to inhibit MGMT and overcome CENU drug resistance. MGMT repairs DNA containing 0-6 methylguanine lesions, as well as CENU-induced lesions, by transfer of the adduct to a 5 aa cysteine-containing acceptor site. Current MGMT inhibitors reveal little about MGMT function, and are weak substrates relative to lesioned DNA. A rational approach to the further development of potent MGMT inhibitors requires identification of those MGMT aa which contribute to the recognition of substrate, both in DNA and as free base, which in turn could guide development of novel inhibitors more closely mimicking the endogenous DNA substrate. We hypothesize that such aa lie outside the MGMT acceptor site, and that specific and limited aa changes outside this region can alter MGMT substrate specificity. We propose to identify such aa by generating a library of MGMT cDNAs containing single point mutations, and following expression of these cDNAs in bacteria, to screen the library for those mutant proteins exhibiting altered substrate specificity. The specific aims of the proposal are: 1) To identify aa which contribute to the MGMT-mediated preferential repair of lesioned DNA versus lesioned free base. 2) To identify aa which contribute to the substrate specificity of MGMT for free bases. 3) To assess the activity and substrate specificity of in vitro derived mutant MGMT proteins in human cells. These studies are likely to contribute to the understanding of how MGMT structure influences the substrate specificity of the protein. This in turn will aid in the development of novel, potent MGMT inhibitors, as well as in the development of predictive assays for MGMT-inhibitor resistance, both of which may contribute to enhanced CENU chemotherapy.